Along with surgery and radiotherapy, chemotherapy continues to be an effective therapy for many cancers. In fact, several types of cancer are now considered to be curable by chemotherapy and include Hodgkin""s disease, large cell lymphoma, acute lymphocytic leukemia, testicular cancer and early stage breast cancer. Other cancers such as ovarian cancer, small cell lung and advanced breast cancer, while not yet curable, are exhibiting positive response to combination chemotherapy.
One of the most important unsolved problems in cancer treatment is drug resistance. After selection for resistance to a single cytotoxic drug, cells may become cross resistant to a whole range of drugs with different structures and cellular targets, e.g., alkylating agents, antimetabolites, hormones, platinum-containing drugs, and natural products. This phenomenon is known as multidrug resistance (MDR). In some types of cells, this resistance is inherent, while in others, such as small cell lung cancer, it is usually acquired.
Such resistance is known to be multifactorial and is conferred by at least two proteins: the 170 kDa P-glycoprotein (MDR1) and the more recently identified 190 kDa multidrug resistance protein (MRP1). Although both MDR1 and MRP1 belong to the ATP-binding cassette superfamily of transport proteins, they are structurally very different molecules and share less than 15% amino acid homology. Despite the structural divergence between the two proteins, by 1994 there were no known consistent differences in the resistance patterns of MDR1 and MRP1 cell lines. However, the association, or lack thereof, of MRP1 and resistance to particular oncolytics is known. See Cole, et. al., xe2x80x9cPharmacological Characterization of Multidrug Resistant MRP-transfected Human Tumor Cellsxe2x80x9d, Cancer Research, 54: 5902-5910, 1994. Doxorubicin, daunorubicin, epirubicin, vincristine, and etoposide are substrates of MRP1, i.e., MRP1 can bind to these oncolytics and redistrubute them away from their site of action, the nucleus, and out of the cell. Id. and Marquardt, D., and Center, M.S., Cancer Research, 52:3157, 1992.
Doxorubicin, daunorubicin, and epirubicin are members of the anthracycline class of oncolytics. They are isolates of various strains of Streptomyces and act by inhibiting nucleic acid synthesis. These agents are useful in treating neoplasms of the bone, ovaries, bladder, thyroid, and especially the breast. They are also useful in the treatment of acute lymphoblastic and myeloblastic leukemia, Wilm""s tumor, neuroblastoma, soft tissue sarcoma, Hodgkin""s and non-Hodgkin""s lymphomas, and bronchogenic carcinoma.
Vincristine, a member of the vinca alkaloid class of oncolytics, is an isolate of a common flowering herb, the periwinkle plant (Vinca rosea Linn). The mechanism of action of vincristine is still under investigation but has been related to the inhibition of microtubule formation in the mitotic spindle. Vincristine is useful in the treatment of acute leukemia, Hodgkin""s disease, non-Hodgkin""s malignant lymphomas, rhabdomyosarcoma, neuroblastoma, and Wilm""s tumor.
Etoposide, a member of the epipodophyllotoxin class of oncolytics, is a semisynthetic derivative of podophyllotoxin. Etoposide acts as a topoisomerase inhibitor and is useful in the therapy of neoplasms of the testis, and lung.
It is presently unknown what determines whether a cell line will acquire resistance via a MDR1 or MRP1 mechanism. Due to the tissue specificity of these transporters and/or in the case where one mechanism predominates or is exclusive, it would be useful to have a selective inhibitor of that one over the other. Furthermore, when administering a drug or drugs that are substrates of either protein, it would be particularly advantageous to co-administer an agent that is a selective inhibitor of that protein. It is, therefore, desirable to provide compounds which are selective inhibitors of MDR1 or MRP1.
The present invention relates to a compound of formula I: 
where:
m is an integer from 1 to 6;
R is COR1, amino, NH-Pg, or NHCOR2;
R1 is hydroxy, C1-C6 alkoxy, or NR3R4;
Pg is an amino protecting group;
R2 is C1-C6 alkyl, substituted C1-C4 alkyl, aryl, substituted aryl, (CH2)n-heterocycle, (CH2)n-substituted heterocycle;
R3 is independently at each occurrence hydrogen or C1-C6 alkyl;
R4 is C1-C6 alkyl, norbornan-2-yl, aryl, substituted aryl, CH2CH(CH3)phenyl, (CH2)nheterocycle, or (CH2)n-substituted heterocycle; and
n is 0, 1, or 2; or a pharmaceutical salt or solvate thereof.
The present invention further relates to a method of inhibiting MRP1 in a mammal which comprises administering to a mammal in need thereof an effective amount of a compound of formula I, or a pharmaceutical salt or solvate thereof.
In another embodiment, the present invention relates to a method of inhibiting a resistant neoplasm, or a neoplasm susceptible to resistance in a mammal which comprises administering to a mammal in need thereof an effective amount of a compound of formula I, or a pharmaceutical salt or solvate thereof, in combination with an effective amount of an oncolytic agent.
The present invention also relates to a pharmaceutical formulation comprising a compound of formula I, or a pharmaceutical salt or solvate thereof, in combination with one or more oncolytics, pharmaceutical carriers, diluents, or excipients therefor.
The current invention concerns the discovery that a select group of compounds, those of formula I, are selective inhibitors of multidrug resistant protein (MRP1) and are thus useful in treating MRP1 conferred multidrug resistance (MDR) in a resistant neoplasm and a neoplasm susceptible to resistance.
The terms xe2x80x9cinhibitxe2x80x9d as it relates to MRP1 and xe2x80x9cinhibiting MRP1xe2x80x9d refer to prohibiting, alleviating, ameliorating, halting, restraining, slowing or reversing the progression of, or reducing MRP1""s ability to redistribute an oncolytic away from the oncolytic""s site of action, most often the neoplasm""s nucleus, and out of the cell.
As used herein, the term xe2x80x9ceffective amount of a compound of formula Ixe2x80x9d refers to an amount of a compound of the present invention which is capable of inhibiting MRP1. The term xe2x80x9ceffective amount of an oncolyticxe2x80x9d refers to an amount of oncolytic capable of inhibiting a neoplasm, resistant or otherwise.
The term xe2x80x9cinhibiting a resistant neoplasm, or a neoplasm susceptible to resistancexe2x80x9d refers to prohibiting, halting, restraining, slowing or reversing the progression of, reducing the growth of, or killing resistant neoplasms and/or neoplasms susceptible to resistance.
The term xe2x80x9cresistant neoplasmxe2x80x9d refers to a neoplasm which is resistant to chemotherapy where that resistance is conferred in part, or in total, by MRP1. Such neoplasms include, but are not limited to, neoplasms of the bladder, bone, breast, lung(small-cell), testis, and thyroid and also includes more particular types of cancer such as, but not limited to, acute lymphoblastic and myeloblastic leukemia, Wilm""s tumor, neuroblastoma, soft tissue sarcoma, Hodgkin""s and non-Hodgkin""s lymphomas, and bronchogenic carcinoma.
A neoplasm which is xe2x80x9csusceptible to resistancexe2x80x9d is a neoplasm where resistance is not inherent nor currently present but can be conferred by MRP1 after chemotherapy begins. Thus, the methods of this invention encompass a prophylactic and therapeutic administration of a compound of formula I.
The term xe2x80x9cchemotherapyxe2x80x9d refers to the use of one or more oncolytics where at least one oncolytic is a substrate of MRP1. A xe2x80x9csubstrate of MRP1xe2x80x9d is an oncolytic that binds to MRP1 and is redistributed away from the oncolytics site of action, (the neoplasm""s nucleus) and out of the cell, thus, rendering the therapy less effective.
The terms xe2x80x9ctreatxe2x80x9d or xe2x80x9ctreatingxe2x80x9d bear their usual meaning which includes preventing, prohibiting, alleviating, ameliorating, halting, restraining, slowing or reversing the progression, or reducing the severity of MRP1 derived drug resistance in a multidrug resistant tumor.
In the general formulae of the present document, the general chemical terms have their usual meanings. For example, the term xe2x80x9cC1-C4 alkylxe2x80x9d refers to methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, cyclobutyl, s-butyl, and t-butyl. The term xe2x80x9cC1-C6 alkylxe2x80x9d refers to a monovalent, straight, branched, or cyclic saturated hydrocarbon containing from 1 to 6 carbon atoms and includes C1-C4 alkyl groups. In addition, C1-C6 alkyl also includes, but is not limited to, cyclopentyl, pentyl, hexyl, cyclohexyl, and the like.
The term xe2x80x9carylxe2x80x9d refers to phenyl, benzyl, and napthyl.
The term xe2x80x9cheterocyclexe2x80x9d refers to a monovalent, saturated, unsaturated, or aromatic mono cyclic or fused ring system of 5 to 7 or 8 to 10 total atoms, respectively, containing from 1 to 3 heteroatoms selected independently from oxygen, sulfur, and nitrogen. Examples of heterocyclic groups include, but are not limited to, furanyl, indolyl, thiophenyl, isoxazolyl, all partially saturated or fully saturated analogues thereof, e.g., tetrahydrofuran, and the like, wherein the point of attachment to the heterocycle is at any position on the ring available for substitution.
The term xe2x80x9csubstituted heterocyclexe2x80x9d refers to a heterocycle ring substituted 1 or 2 times independently with a C1-C6 alkyl, halo, benzyl, phenyl, trifluoromethyl, or an oxo group.
The term xe2x80x9csubstituted arylxe2x80x9d refers to a phenyl, benzyl, and napthyl group, respectively, substituted from 1 to 5 times independently with halo, hydroxy, trifluoromethyl, N(R1)2, NH-Pg, C1-C4 alkoxy, benzyloxy, CO2R1, SO2NH2, trifluoromethoxy, or nitro.
The term xe2x80x9csubstituted C1-C4 alkylxe2x80x9d refers to a C1-C4 alkyl group where 1 to 3 hydrogens have been replaced by the same halide, e.g., trifluoromethyl.
The term xe2x80x9chaloxe2x80x9d or xe2x80x9chalidexe2x80x9d refers to fluoro, chloro, bromo, or iodo.
The term xe2x80x9cC1-C4 alkoxyxe2x80x9d refers to a C1-C4 alkyl group attached through an oxygen atom.
The term xe2x80x9camino protecting groupxe2x80x9d as used in this specification refers to a substituent(s) of the amino group commonly employed to block or protect the amino functionality while reacting other functional groups on the compound. Examples of such amino-protecting groups include the formyl group, the trityl group, the phthalimido group, the acetyl group, the trichloroacetyl group, the chloroacetyl, bromoacetyl, and iodoacetyl groups, urethane-type blocking groups such as benzyloxycarbonyl, 9-fluorenylmethoxycarbonyl (xe2x80x9cFMOCxe2x80x9d), and the like; and like amino protecting groups. The species of amino protecting group employed is not critical so long as the derivatize amino group is stable to the condition of subsequent reaction(s) on other positions of the molecule and can be removed at the appropriate point without disrupting the remainder of the molecule. Similar amino protecting groups used in the cephalosporin, penicillin, and peptide arts are also embraced by the above terms. Further examples of groups referred to by the above terms are described by T. W. Greene, xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d, John Wiley and Sons, New York, N.Y., 1991, Chapter 7 hereafter referred to as xe2x80x9cGreenexe2x80x9d. A preferred amino protecting group is t-butyloxycarbonyl.
The term xe2x80x9ccarbonyl activating groupxe2x80x9d refers to a substituent of a carbonyl that renders that carbonyl prone to nucleophilic addition. Suitable activating groups are those which have a net electron withdrawing effect on the carbonyl. Such groups include, but are not limited to, alkoxy, aryloxy, nitrogen containing aromatic heterocycles, or amino groups such as oxybenzotriazole, imidazolyl, nitrophenoxy, pentachlorophenoxy, N-oxysuccinimide, N,Nxe2x80x2-dicyclohexylisoure-O-yl, N-hydroxy-N-methoxyamino, and the like; acetates, formates, sulfonates such as methanesulfonate, ethanesulfonate, benzenesulfonate, or p-toluenylsulfonate, and the like; and halides especially chloride, bromide, or iodide.
In general, the term xe2x80x9cpharmaceuticalxe2x80x9d when used as an adjective means substantially non-toxic to living organisms. For example, the term xe2x80x9cpharmaceutical saltxe2x80x9d as used herein, refers to salts of the compounds of formula I which are substantially non-toxic to living organisms. See, e.g., Berge, S. M, Bighley, L. D., and Monkhouse, D. C., xe2x80x9cPharmaceutical Saltsxe2x80x9d, J. Pharm. Sci., 66:1, 1977. Typical pharmaceutical salts include those salts prepared by reaction of the compounds of formula I with an inorganic or organic acid or base. Such salts are known as acid addition or base addition salts respectively. These pharmaceutical salts frequently have enhanced solubility characteristics compared to the compound from which they are derived, and thus are often more amenable to formulation as liquids or emulsions.
Examples of pharmaceutical acid addition salts are the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, xcex3-hydroxybutyrate, glycollate, tartrate, methanesulfonate, ethanesulfonate, propanesulfonate, naphthalene-1-sulfonate, napththalene-2-sulfonate, mandelate, and the like of a compound of formula I.
Examples of pharmaceutical base addition salts are the ammonium, lithium, potassium, sodium, calcium, magnesium, methylamino, diethylamino, ethylene diamino, cyclohexylamino, and ethanolamino salts, and the like of a compound of formula I.
The term xe2x80x9csolvatexe2x80x9d represents an aggregate that comprises one or more molecules of the solute, such as a formula I compound, with one or more molecules of solvent.
The term xe2x80x9csuitable solventxe2x80x9d refers to a solvent which is inert to the ongoing reaction and sufficiently solubilizes the reactants to effect the desired reaction. Examples of suitable solvents include but are not limited to, dichloromethane, chloroform, 1,2-dichloroethane, diethyl ether, acetonitrile, ethyl acetate, 1,3-dimethyl-2-imidazolidinone, tetrahydrofuran, dimethylformamide, toluene, chlorobenzene, dimethylsulfoxide, mixtures thereof, and the like.
The term xe2x80x9ccarbonyl activating reagentxe2x80x9d refers to a reagent that converts the carbonyl of a carboxylic acid group to one that is more prone to nucleophilic addition and includes, but is not limited to, such reagents as those found in xe2x80x9cThe Peptidesxe2x80x9d, Gross and Meienhofer, Eds., Academic Press (1979), Ch. 2 and M. Bodanszky, xe2x80x9cPrinciples of Peptide Synthesisxe2x80x9d, 2nd Ed., Springer-Verlag Berlin Heidelberg, 1993, hereafter referred to as xe2x80x9cThe Peptidesxe2x80x9d and xe2x80x9cPeptide Synthesisxe2x80x9d respectively. Specifically, carbonyl activating reagents include nucleophilic sources of a halogen such as, thionyl bromide, thionyl chloride, oxalyl chloride, and the like; alcohols such as nitrophenol, pentachlorophenol, and the like; amines such as N-hydroxy-N-methoxyamine and the like; acid halides such as acetic, formic, methanesulfonic, ethanesulfonic, benzenesulfonic, or p-tolenesulfonic acid halide, and the like; and compounds such as 1,1xe2x80x2-carbonyldiimidazole, benzotriazole, imidazole, N-hydroxysuccinimide, dicyclohexylcarbodiimide, and the like.
The term xe2x80x9csuitable thermodynamic basexe2x80x9d refers to a base which acts as a proton trap for any protons which may be produced as a byproduct of the desired reaction or to a base which provides a reversible deprotonation of an acidic substrate and is reactive enough to effect the desired reaction without significantly effecting any undesired reactions. Examples of thermodynamic bases include, but are not limited to, carbonates, bicarbonates, and hydroxides (e.g., lithium, sodium, or potassium carbonate, bicarbonate, or hydroxide), tri-(C1-C4 alkyl)amines, or aromatic nitrogen containing heterocycles (e.g., pyridine).
While all of the compounds of the present invention are useful, certain of the compounds are particularly interesting and are preferred. The following listing sets out several groups of preferred compounds, formulations, and methods. It will be understood that each of the listings may be combined with other listings to create additional groups of preferred embodiments.
a) m is an integer from 2 to 6;
b) R is amino;
c) R is t-butyloxycarbonylamino;
d) R is trifluoroacetylamino;
e) R is 3,4,5-trimethoxybenzoylamino;
f) R is carboxy;
g) R is 3,4,5-trimethoxyanilinylcarboxy;
h) R is 3,4,5-trimethoxybenzylaminylcarboxy;
i) The compounds of the Examples section;
j) The compound is a pharmaceutical salt;
k) The compound is the hydrochloride salt;
l) The method where the mammal is a human;
m) The method where the oncolytic(s) is selected from: doxorubicin, daunorubicin, epirubicin, vincristine, and etoposide;
n) The method where the neoplasm is of the Wilm""s type, bladder, bone, breast, lung(small-cell), testis, or thyroid or the neoplasm is associated with acute lymphoblastic and myeloblastic leukemia, neuroblastoma, soft tissue sarcoma, Hodgkin""s and non-Hodgkin""s lymphomas, or bronchogenic carcinoma; and
o) The formulation where the oncolytic(s) is selected from the group: doxorubicin, daunorubicin, epirubicin, vincristine, and etoposide.
The compounds of the present invention can be prepared by a variety of procedures, some of which are illustrated in the Schemes below. The particular order of steps required to produce the compounds of formula I is dependent upon the particular compound being synthesized, the starting compound, and the relative lability of the substituted moieties.
Compounds of formula I may be prepared from compounds of formula II as illustrated in Scheme 1 below where R and m are as described supra. 
Compounds of formula I may be prepared by dissolving or suspending a compound of formula II in a suitable solvent and adding a suitable thermodynamic base. Typically a preferred and convenient solvent is dimethylformamide. Usually a convenient and preferred thermodynamic base is sodium hydroxide added as a 2N solution in methanol. The reactants are typically combined at room temperature and the resulting solution is typically allowed to react for from 30 minutes to about 18 hours. Preferably, the mixture is stirred about 1 to about 10 hours, and is most preferably stirred for about 3.5 hours to about 7 hours. The base is typically employed in a large molar excess, usually in about a 4 to about an 8 molar excess relative to the compound of formula II. Preferably, about a 5 to about a 7 molar excess is typically employed. Certain intermediates, discussed below, of compounds of formula I may also be prepared by the method discussed above.
Any hydroxy or amino protecting groups found in the cyclized compound of formula I may optionally be removed as taught in Greene to provide the free amino or free hydroxy compounds of formula I. Preferred choices of protecting groups and methods for their removal may be found in the Preparations and Examples sections below.
Compounds of formula I, where R is COR1 may also be prepared from compounds of formula I(a) as illustrated in Scheme 2 below where R5 is a carboxy activating group, R6 is C1-C6 alkoxy, or NR3R4 and m is as described supra. 
Compounds of formula I(a), prepared as described in Scheme 1, may be converted to other compounds of the invention. For example, acids of formula I(a) may be activated to form the activated carboxylic acids of formula III by methods well known in the chemical arts. See, e.g., The Peptides, Peptide Synthesis, and the Examples and Preparations sections below.
Compounds of formula I(b) may then be prepared by dissolving or suspending a compound of formula III in a suitable solvent, optionally in the presence of a suitable thermodynamic base, and adding an amine of formula IV. Typically a preferred and convenient solvent is dichloromethane. Preferred bases are triethylamine and piperidinylmethylpolystyrene resin. The amine is typically employed in a molar excess. For example, about a 1.5 to about a 3 molar excess, relative to the compound of formula III, is usually employed. About 1.8 to about 2.2 molar excess is typically preferred. The reaction is usually performed in a temperature range of about 0xc2x0 C. to about the reflux temperature of the solvent for from 10 minutes to 18 hours. Preferably, the reaction is performed at about 15xc2x0 C. to about 40xc2x0 C. for 5 minutes to about 2.5 hours.
Alternatively, the compound of formula I(a) may be activated and the addition of a compound of formula IV may be performed in a one pot process as described in Examples 26 and 38 below.
The compounds of formula I(b) where R6 is C1-C6 alkoxy, i.e., esters, may be prepared by methods very well known in the chemical arts. For instruction on the conversion of activated carboxylic acids to esters see, e.g., Larock, xe2x80x9cComprehensive Organic Transformationsxe2x80x9d, pgs. 978-979, VCH Publishers, New York, N.Y., 1989, hereafter referred to as xe2x80x9cLarockxe2x80x9d. Alternatively, these compounds of formula I(b) may be prepared directly from the acids of formula I(a) as taught in Larock at pages 966-972.
Compounds of formula I where R is amino or NHCOR2 may be prepared from compounds of formula I(c) as illustrated in Scheme 3 below where m, Pg, R2, and R5 are as described supra. 
Compounds of formula I(c) may be converted to other compounds of the invention. For example, a compound of formula I(c) may have its protecting group removed as taught in Greene or in the Examples section which follows to form a compound of formula I(d). These compounds of formula I(d) may then be converted to other compounds of the invention as well. For example, a compound of formula I(d) dissolved or suspended in a suitable solvent, optionally in the presence of a thermodynamic base, may be treated with a compound of formula VI to provide a compound of formula I(e). Typically a preferred and convenient solvent is dimethylformamide or a mixture of dichloromethane and dimethylformamide. When a base is employed, triethylamine or N-methylmorpholine is typically a preferred base. Furthermore, when a base is employed, the base and compound of formula VI are typically employed in a stoichiometric to large molar excess. For example a 1.0 to 4 molar excess, relative to the compound of formula I(d), is generally employed. When a base is not employed, the compound of formula VI is typically employed in a relatively larger stoichiometric excess. It is preferred to perform the reaction in the presence of a base with from about 1.8 to about 2.2 equivalents of a compound of formula VI. The reaction is usually performed at a temperature range of about 0xc2x0 C. to about the reflux temperature of the solvent for from 10 minutes to 24 hours. Preferably, the reaction is performed at about 15xc2x0 C. to about 40xc2x0 C. for from 4 to about 18 hours.
The pharmaceutical salts of the invention are typically formed by reacting a compound of formula I with an equimolar or excess amount of acid or base. The reactants are generally combined in a mutual solvent such as diethylether, tetrahydrofuran, methanol, ethanol, isopropanol, benzene, and the like for acid addition salts, or water, an alcohol or a chlorinated solvent such as dichloromethane for base addition salts. The salts normally precipitate out of solution within about one hour to about ten days and can be isolated by filtration or other conventional methods.
Acids commonly employed to form pharmaceutical acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulfonic, methanesulfonic acid, ethanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, tartaric acid, benzoic acid, acetic acid, and the like. Preferred pharmaceutical acid addition salts are those formed with mineral acids such as hydrochloric acid, hydrobromic acid, and sulfuric acid, and those formed with organic acids such as maleic acid, tartaric acid, and methanesulfonic acid.
Bases commonly employed to form pharmaceutical base addition salts are inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Such bases useful in preparing the salts of this invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate, and the like. The potassium and sodium salt forms are particularly preferred.
It should be recognized that the particular counterion forming a part of any salt of this invention is not of a critical nature, so long as the salt as a whole is pharmacologically acceptable and as long as the counterion does not contribute undesired qualities to the salt as a whole.
The starting materials for the processes of the present invention may be obtained by a number of routes. For example, compounds of formula II, I(a), and I(c) may be prepared according to the route shown in Scheme 4 where m and R are as described supra. 
Compounds of formula II may be prepared by dissolving or suspending a compound of formula VII in a suitable solvent and adding a compound of formula VIII and a suitable thermodynamic base. Dichloromethane is a convenient solvent and is typically preferred. Triethylamine is usually a preferred thermodynamic base. This amide forming reaction is also preferably run in the presence of dimethylamino pyridine (DMAP). The compound of formula VIII is typically and preferably employed in an equimolar amount, relative to the compound of formula VII, but a slight excess (about a 0.05 to about 0.15 molar excess) is acceptable. The thermodynamic base is typically employed in a slight molar excess. For example, about a 1.01 to about a 1.2 molar excess, relative to the compound of formula VII, is typically employed. About a 1.05 to about 1.15 molar excess is generally preferred. The DMAP is employed in a catalytic fashion. For example, about 5 molar percent to about 15 molar percent, relative to the compound of formula VII, is typically employed. A 10 molar percent is usually preferred.
Although the transformations described in Schemes 2 and 3 may be performed before the cyclization described in Scheme 1 to provide the compounds of formula VII with a fully elaborated R substituent, it is preferred to perform these reactions after the cyclization. Thus, preferred starting materials for the reaction of Scheme 1 and 4 are the compounds of formula II and VII where R is NH-Pg or COR1 where R1 is C1-C6 alkoxy. Furthermore, if the reaction of Scheme 1 is performed with a compound of formula II where R is COR1 and R1 is C1-C6 alkoxy, under the conditions described for the cyclization, that ester will be cleaved to the acid, i.e., will give the compounds of formula I(a).
Compounds of formula IV, V, VI, VII, and VIII are known in the art and, to the extent not commercially available, are readily synthesized by standard procedures commonly employed in the art.
The optimal time for performing the reactions of Schemes 1-4 can be determined by monitoring the progress of the reaction via conventional chromatographic techniques. Furthermore, it is preferred to conduct the reactions of the invention under an inert atmosphere, such as, for example, argon, or, particularly, nitrogen. Choice of solvent is generally not critical so long as the solvent employed is inert to the ongoing reaction and sufficiently solubilizes the reactants to effect the desired reaction. The compounds of formula I(d) and II are preferably isolated and purified before their use in subsequent reactions. These compounds may crystallize out of the reaction solution during their formation and then be collected by filtration, or the reaction solvent may be removed by extraction, evaporation, or decantation. These intermediate and final products of formula I may be further purified, if desired by common techniques such as recrystallization or chromatography over solid supports such as silica gel or alumina.
The following Preparations and Examples are provided to better elucidate the practice of the present invention and should not be interpreted in any way as to limit the scope of same. Those skilled in the art will recognize that various modifications may be made while not departing from the spirit and scope of the invention. All publications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. The terms and abbreviations used in the instant Preparations and Examples have their normal meanings unless otherwise designated. For example xe2x80x9cxc2x0C.xe2x80x9d, xe2x80x9cNxe2x80x9d, xe2x80x9cmmolxe2x80x9d, xe2x80x9cgxe2x80x9d, xe2x80x9cmLxe2x80x9d, xe2x80x9cMxe2x80x9d, xe2x80x9cIRxe2x80x9d, xe2x80x9cMS(FD)xe2x80x9d,and xe2x80x9cMS(IS)xe2x80x9d refer to degrees Celsius, normal or normality, millimole or millimoles, gram or grams, milliliter or milliliters, molar or molarity, infra red spectrometry, field desorption mass spectrometry, and ion spray mass spectrometry respectively. In addition, the absorption maxima listed for the IR spectra are only those of interest and not all of the maxima observed.
N-t-butyloxycarbonyl-1,2-diaminoethane (355 mg, 2.22 mmol), dimethylaminopyridine (27 mg, 0.222 mmol), and triethylamine (247 mg, 2.44 mmol) were combined in 5 mL of dry dichloromethane. 3-(2-Chloro-6-fluorophenyl)-5-methylisoxazol-4-oyl chloride (607 mg, 2.22 mmol) was added in slowly. The reaction was stirred for 3 hours at room temperature under nitrogen. The reaction was diluted with ethyl acetate and water. The ethyl acetate was separated and washed three times each with 25 mL of 1N aqueous hydrochloric acid and brine, dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was treated with hexanes to crystallize 794 mg of the title compound. (90%). EA calculated for: C18H21N3O4ClF: C, 54.34; H, 5.32; N, 10.56. Found: C, 54.30; H, 5.36; N, 10.44. MS(IS) m/z 398 (M+).
N-t-butyloxycarbonyl-1,3-diaminopropane (574 mg, 3.29 mmol) and 3-(2-chloro-6-fluorophenyl)-5-methylisoxazol-4-oyl chloride (903 mg, 3.29 mmol) were converted to 1.36 g of the title compound by the procedure of Preparation 1. (100%). EA calculated for: C19H23N3O4ClF: C, 55.41; H, 5.63; N, 10.20. Found: C, 55.17; H, 5.71; N, 9.99. MS(IS) m/z 412 (M+).
N-t-butyloxycarbonyl-1,4-diaminobutane (985 mg, 5.23 mmol) and 3-(2-chloro-6-fluorophenyl)-5-methylisoxazol-4-oyl chloride (1.43 g, 5.23 mmol) were converted to 2.23 g of the title compound by the procedure of Preparation 1 except that the reaction time was about 18 hours. (100%). EA calculated for: C20H25N3O4ClF: C, 56.41; H, 5.92; N, 9.87. Found: C, 56.12; H, 5.75; N, 9.67. MS(FD) m/z 426 (M+).
N-t-butyloxycarbonyl-1,5-diaminopentane (1.04 g, 5.14 mmol) and 3-(2-chloro-6-fluorophenyl)-5-methylisoxazol-4-oyl chloride (1.41 g, 5.14 mmol) were converted to 2.14 g of the title compound by the procedure of Preparation 1. (95%). MS(IS) m/z 412 (M+). IR(CHCl3) 3496, 3012, 2936, 1708, 1663, 1611, 1510 cmxe2x88x921.
N-t-butyloxycarbonyl-1,6-diaminohexane hydrochloride salt (1.02 g, 4.03 mmol) and 3-(2-chloro-6-fluorophenyl)-5-methylisoxazol-4-oyl chloride (1.11 g, 4.03 mmol) were converted to 1.84 g of the title compound by the procedure of Preparation 1. (100%). EA calculated for: C22H29N3O4ClF: C, 58.21; H, 6.44; N, 9.26. Found: C, 57.97; H, 6.20; N, 9.43. MS(IS) m/z 454 (M+)
N-t-butyloxycarbonyl-4-aminobutanoic acid (1 g, 4.92 mmol) was dissolved in 10 mL of a freshly prepared mixture of hydrochloric acid and methanol (2.5 mL of acetyl chloride in 35 mL of methanol). The reaction was stirred for about 2 hours and then stripped down. The residue was taken up in ethyl acetate, washed three times each with aqueous sodium bicarbonate and brine, dried over sodium sulfate, and filtered and concentrated down leaving 708 mg of the title compound. (94%). EA calculated for C5H12ClNO2: C, 39.10; H, 7.87; N, 9.12. Found: C, 38.89; H, 7.65; N, 8.94. MS(FD) m/z 118 (M+for free amine).
N-t-butyloxycarbonyl-5-aminopentanoic acid (1 g, 4.60 mmol) was converted to the title compound by the procedure of Preparation 6 to give 705 mg. (91%). EA calculated for C6H14ClNO2: C, 42.99; H, 8.42; N, 8.36. Found: C, 43.02; H, 8.15; N, 8.18. MS(FD) m/z 132 (M+for free amine).
N-t-butyloxycarbonyl-6-aminohexanoic acid ;500 mg, 2.16 mmol) was converted to the title compound by the procedure of Preparation 6 to give 357 mg. (91%). EA calculated for C7H16ClNO2: C, 46.28; H, 8.88; N, 7.71. Found: C, 46.04; H, 8.88; N, 7.51. MS(FD) m/z 146 (M+for free amine).
N-t-butyloxycarbonyl-7-aminoheptanoic acid (1 g, 4.08 mmol) was converted to the title compound by the procedure of Preparation 6 to give 742 mg. (93%). EA calculated for C8H17ClNO2: C, 49.10; H, 9.27; N, 7.16. Found: C, 49.10; H, 9.18; N, 7.03. MS(FD) m/z 160 (M+for free amine).
Methyl-4-aminobutanoate hydrochloride (668 mg, 4.34 mmol) and 3-(2-chloro-6-fluorophenyl)-5-methylisoxazol-4-oyl chloride (1.19 g, 4.35 mmol) were converted to 1.60 g of the title compound by the procedure of Preparation 1 except that the residue was not treated with hexanes. (100%). EA calculated for: C16H16N2O4ClF: C, 54.17; H, 4.55; N, 7.90. Found: C, 54.41; H, 4.58; N, 7.78. MS(FD) m/z 355 (M+).
Methyl-5-aminopentanoate hydrochloride (694 mg, 4.14 mmol) and 3-(2-chloro-6-fluorophenyl)-5-methylisoxazol-4-oyl chloride (1.24 g, 4.52 mmol) were converted to 1.36 g of the title compound by the procedure of Preparation 10. (100%). EA calculated for: C17H18N2O4ClF: C, 55.37; H, 4.92; N, 7.60. Found: C, 55.44; H, 5.00; N, 7.52. MS(FD) m/z 368 (M+).
Methyl-6-aminohexanoate hydrochloride (349 mg, 1.92 mmol) and 3-(2-chloro-6-fluorophenyl)-5-methylisoxazol-4-oyl chloride (527 mg, 1.92 mmol) were converted to 2.23 g of the title compound by the procedure of Preparation 10. (100%). EA calculated for: C18H20N2O4ClF: C, 56.48; H, 5.27; N, 7.32. Found: C, 56.66; H, 5.20; N, 7.07. MS(FD) m/z 382.2 (M+).
Methyl-7-aminoheptanoate hydrochloride (733 mg, 3.75 mmol) and 3-(2-chloro-6-fluorophenyl)-5-methylisoxazol-4-oyl chloride (1.03 g, 3.75 mmol) were converted to 1.48 g of the title compound by the procedure of Preparation 10. (100%). EA calculated for: C19H22N2O4ClF: C, 57.51; H, 5.59; N, 7.06. Found: C, 57.78; H, 5.70; N, 6.80. MS(FD) m/z 396.1 (M+).